Each year, several hundred million people are infected with P. falciparum, which causes the most severe form of malaria in humans leading to 1 to 2 million deaths [Marti M et al., Science. 2004, 306 (5703):1930-3]. The primary chemotherapeutic drugs like Chloroquine and Pyrimethamine are of little use because parasite has developed resistance against them [V. E. Rosario, Nature, 261, 1976, p 585]. Recent reports suggest that resistance to Artemisinin, the only effective anti-malarial drug at present, is now emerging [Dondorp A M et al., N. Engl. J. Med. 361, 455 (2009), Noedl H et al., N. Engl. J. Med. 359, 2619 (2008)]. Therefore, the search for new drugs must continue. In the quest for new drugs, it is also important to revisit the efficacies of some of the drugs whose potential has not been verified in depth for anti-malarial activities. Acriflavin, a mixture of 3,6-diamino-10-methylacridinum chloride (Trypflavin) and 3,6-diaminoacridine (Proflavin) was developed in 1912 by German medical researcher Paul Ehrlich [Wainwright M, J. Antimicrobial Chemothererapy, 2001, 47, 113]. Acriflavin is an anti-bacterial Acridine and it has been used widely as a topical antiseptic (Browning C H et al., Br Med J. 1917, 2 (2951):70-5]. Besides anti-microbial actions, Acriflavin has been recently shown to have potential anti-cancer activity in mice [Lee K et al., Proc Natl Acad Sci USA. 2009; 106 (42):17910-5]. Before the invention of Chloroquine, it was used as anti-malarial. Although Acriflavin had a potential to be used as anti-malarial, the anti-malarial activity of Acriflavin was not studied further in details.
A possible target for antibacterial activity of Acriflavin is DNA topoisomerase/bacterial gyrase. Gyrase is a type II topoisomerase with two subunits (A and B), essential for relieving the positive supercoiling that may arise ahead of replication fork or due to transcription. Gyrase is not only capable of relieving positive supercoiling, it can also introduce negative supercoiling that is the preferred state of bacterial circular chromosome. A mutation in the gyrase B gene (acrB) has been shown to be responsible for making E. coli susceptible to Acriflavin (Funatsuki, K et al., JBC, 1997, 272, 13302-08). It was further shown that the DNA binding activity of gyrase enzyme with the acrB mutation was affected in the presence of Acriflavin. These results indicated that gyrase could be a possible target for Acriflavin.
Interestingly, the human malarial parasite P. falciparum contains both the subunits of bacterial gyrase essential for the replication and maintenance of apicoplast organelle. Apicoplast has been acquired by the parasites by endosymbiotic pathways thereby making it susceptible to many drugs that target bacterial replication and transcription machinery [Goodman C D, et al., Mol Biochem Parasitol. 2007 April; 152 (2):181-91]. Interestingly, analysis of the gyrase B amino acid sequences from P. falciparum and E. coli reveal the presence of similar residues around the acrB mutation region. The residue Arg (760) found in acrB mutant E. coli strain is identical in PfGyrB Arg (965).
The presence of bacterial type gyrase in Plasmodium prompted to investigate the potency of Acriflavin as anti-malarial agent and its mechanism of action. Acriflavin is a FDA approved drug used in clinical trail against cancer with no or minimal toxicity. It is found that Acriflavin not only kills chloroquine sensitive and resistant malaria parasites in vitro in nano molar range, it also suppresses parasite growth significantly in mouse model. Interestingly, it is found that Acriflavin is accumulated specifically in the infected erythrocytes and not in the uninfected erythrocytes. Further, it was found that Acriflavin affects Plasmodium gyrase activity in vitro. It remains to be seen further whether gyrase is a target of Acriflavin in vivo too. These findings establish Acriflavin as a potent anti-malarial both in vitro and in vivo that may have far-reaching consequences in the quest of new anti-malarial drugs.
In the present invention, it is shown that Acriflavin is a potent anti-malarial both in vivo and in vitro with IC50 value residing within nanomolar range. Moreover, this inhibition seems mediated through specific accumulation of Acriflavin in the parasites within the infected RBC only. Acriflavin has been known for its trypanocidal, antibacterial and antiviral activities. The effect of Acriflavin on cancer cells has also been reported [Lee K et al., 2009, PNAS, 106 (42):17910-5]. Acriflavin can inhibit the tumor growth in mice possibly through affecting the dimerization of hypoxia inducible factor HIF-1 that plays important role in cancer progression [Lee K et al., 2009, PNAS, 106 (42):17910-5]. These results indeed support the rationale of using Acriflavin in various diseases. Although there is a concern that Acriflavin is a DNA intercalating agent, administration of Acriflavin in patients over five months does not cause any major side effects suggesting the potential use of Acriflavin in clinical trials.
It is found that Acriflavin inhibits Plasmodium gyrase activity that is essential for apicoplast DNA replication. It is possible that Acriflavin may have multiple targets. In vitro, acridine derivatives inhibit Topoisomerase II activity and affects hematin formation that may be crucial for haeme detoxification [Auparakkitanon S and Wilairat P., Biochem Biophys Res Commun 2000, 269 (2):406-9; Auparakkitanon S et al. Antimicrob Agents Chemother. 2003, 47 (12):3708-12] Multiple targets lower the possibility of rapid incidence of drug resistance.
The perception and some evidences related to the DNA interacting property of Acridine ring containing compounds go against its widespread use (Lerman L S, PNAS, 1963, Jan. 15; 49: 94-102). In E. coli, higher rate of cell death, mutation frequency and blockage of DNA, RNA and protein synthesis takes place following UV exposure of cells in the presence of micromolar level of Acriflavin (1 μg/ml=3.8 μM). [Doudney C. O., Biochem Biophys Res Commun 1964, 15 (1):70-5]. The same study also reports no measurable incidence of mutation in non-UV exposed Acriflavin treated cells. It has been suggested that Acriflavin possibly interacts with UV damage site (thymine dimer, which is otherwise repairable) leading to the increased lethality and mutation rate. These results indicate that Acriflavin may not be mutagenic by itself. However, the exposure to UV light may affect the cells. Since the IC50 value for effective killing of the parasites is within nanomolar range, the concerns over the DNA intercalating and DNA damaging activity of Acriflavin may be over-speculative. The efficient uptake and retention of Acriflavin by the parasites may also add to the potent anti-malarial effect of Acriflavin.
Taken together, it was demonstrated convincingly that Acriflavin shows potent antimalarial activity in both in vitro and in vivo working in the nanomolar range. Moreover, Acriflavin is accumulated specifically in the infected RBC containing parasites and not in the uninfected RBC. Further, it is shown that gyrase is a potential target of Acriflavin in vitro. As per knowledge to date, in vitro and in vivo anti-plasmodial activity of Acriflavin has not been reported so far which makes it a candidate for anti-malarial drug.